Enzyme catalysts have a myriad of existing and potential applications. Various techniques for creating new and improved enzyme variants are now available, allowing for the creation of large libraries of mutants or variants that can be screened for desired properties. For example, directed evolution has been successfully applied to improve a variety of enzyme properties, including substrate specificity, activity in organic solvents, and stability at high temperatures, which are often critical for industrial applications (Arnold, F. H. Accounts Chem. Res. 31, 125 (1998)). The directed evolution approach uses DNA shuffling for simultaneous random mutagenesis and recombination to generate a variant having an improved desirable property over the existing wild-type protein. Point mutations can be generated, for example, using the intrinsic infidelity of Taq-based polymerase chain reactions (PCR) associated with reassembly of nucleic acid sequences. In one example, Stemmer and coworkers applied this technique to the gene encoding for green fluorescence protein (GFP), which resulted in a protein that folded better than the wild type in E. coli (Crameri, A.; et al. Nature Biotechnol. 14, 315 (1996)). However, the need for new enzymes having new or enhanced biological properties remains. For example, to date, there are no known enzymes that selectively oxidize the 6-hydroxyl group of D-glucose. Such enzymes would be useful, particularly for chemical synthesis applications.
Galactose oxidase (D-galactose: oxygen 6-oxidoreductase, GAO), designated EC 1.1.3.9 by the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology, is an oxidation enzyme containing a single copper ion. GAO is secreted by a number of fungal species, but no bacterium has been reported to produce the enzyme (Aisaka, K., and Terada, O., (1981), Agric. Biol. Chem., 45(10), 2311–2316).
GAO from one particular fungal species, Fusarium NRRL 2903, formerly known as Dactylium dendroides, has been extensively studied (Borman, C. D. et al. (1997) J. Biol. Inorg. Chem. 2,480–487). Fusarium GAO is a glycoprotein with a carbohydrate content of about 1.7% and consists of a single polypeptide chain of 639 amino acid residues with a molecular mass of 68 kD (the cDNA sequence is depicted in SEQ ID NO:1, which corresponds to nucleotide residues 962 to 2878 of GenBank Accession No. M86819; and the amino acid sequence in SEQ ID NO: 2, which corresponds to amino acid residues 42 to 680 of GenBank accession No. AAA16228.1 (Mendonca, M. H., and Zancan, G. T. (1987) Arch. Biochem. Biophys. 272, 507–514; Ito, N., et al. (1994) J. Mol. Biol. 238, 794–81477).
The reaction catalyzed by galactose oxidase (GAO) is the oxidation of primary alcohols to the corresponding aldehydes, coupled to the two-electron reduction of O2 to hydrogen peroxide (Whitaker, M. W., and Whitaker, J. W. (1988) J. Biol. Chem. 263, 6074–6080). See FIG. 1. Galactose oxidase (GAO) is capable of oxidizing the hydroxyl group of the sixth carbon of D-galactose. The kinetic parameters of GAO for the oxidation of galactose are: Km=67 mM, kcat=3,000 sec−1, kcat/Km=45×103 M−1sec−1 (Baron, A. J. (1994) J. Biol. Chem. 269, 25095–25105). GAO exhibits prochiral specificity (only the pro-S hydrogen is abstracted) as well as enantiomeric specificity for galactose (only D-galactose is oxidized by the enzyme) (Avigad, G., et al. (1962) J. Biol. Chem. 237, 2736–2743, Maradufu, A., et al. (1971) Canad. J. Chem. 49, 3429–3436).
In addition to D-galactose, GAO oxidizes a broad range of substrates. For example, GAO also accepts alpha- and beta-galactopyranosides, oligo- and polysaccharides and considerably smaller molecules, such as glycerol and allyl alcohol, as well as many other kinds of sugars and primary alcohols. However, in spite of the broad substrate specificity of GAO, it discriminates against D-glucose, the C-4 epimer of D-galactose, as a substrate or ligand. D-glucose does not bind to GAO, even at concentrations as high as 1M (Avigad, G., et al. (1962) J. Biol. Chem. 237, 2736–2743; Wachter, R. M., and Branchaud, B. L. (1996) J. Am. Chem. Soc. 118, 2782–2789).
GAO has one copper (II) ion associated with its active site and related to its oxidation properties. The GAO enzyme has three predominantly beta-structure domains (Ito, N., et al. (1991) Nature 350, 87–90). The copper ion lies on the solvent-accessible surface of the second and largest domain (residues 156–532) (Ito, N., et al. (1994) J. Mol. Biol. 238, 794–814; Ito, N., et al. (1995) Methods Enzymol. 258, 235–262). Tyr-272, Tyr-495, His-496, His-581 and a water molecule are the copper ligands at pH 7.0. The crystal structure also reveals a novel thioether bond linking Cys-228 and Tyr-272 and supports the presence of a tyrosine free radical at the active site (Whitaker, M. W., and Whitaker, J. W. (1988) J. Biol. Chem. 263, 6074–6080). A 3-D model of GAO and its active site structure is shown in FIG. 2.
Structure and amino acid residues related to GAO catalysis have been characterized and reported (Borman, C. D., et al. (1997) J. Biol. Inorg. Chem. 2, 480–487; Ito, N., et al. (1994) J. Mol. Biol. 238, 794–814; Wachter, R. M., and Branchaud, B. L. (1996) J. Am. Chem. Soc. 118, 2782–2789; Baron, A. J., et al. (1994) J. Biol. Chem. 269, 25095–25105; Ito, N., et al. (1991) Nature 350, 87–90; Reynolds, M. P., et al. (1997) J. Biol. Inorg. Chem. 2, 327–335; McPherson, M. J., et al. (1993), Biochem. Soc. Transact., 21, 752–756. (See also FIG. 2). For example, site-directed mutagenesis of Tyr-495 and Cys-228 have confirmed their involvement in galactose oxidation (Baron, A. J., et al. (1994) J. Biol. Chem. 269, 25095–25105; Reynolds, M. P., et al. (1997) J. Biol. Inorg. Chem. 2, 327–335). In addition, models illustrating residues involved in galactose binding have been proposed. For example, tryptophan 290 (W290) has been identified as a component of the free radical site of galactose oxidase (Baron, A. J., et al. (1994) J. Biol. Chem. 269, 25095–25105). The crystal structure also demonstrated that four residues, (histidine 496 and 581 (H496 and H581) and tyrosine 272 and 495 (Y272 and Y495) coordinate the cooper ion of galactose oxidase (Ito, N., et al. (1994) J. Mol. Biol. 238, 794–814). A pocket containing phenylalanines 194 and 464 (F194 and F464) interacts with the D-galactose backbone. See FIG. 2. These models may explain why galactose oxidase cannot oxidize D-glucose to any significant extent. For example, it has been hypothesized that arginine 330 (R330) may be important for hydrogen bonding of GAO and 0–4 of D-galactose, and that steric hindrance may prevent this bond forming between GAO and glucose (Ito, N., et al. (1991) Nature 350, 87–90).
For general literature describing GAO, its structure, and its enzyme activity, see Bibliography section entitled “GAO Literature.”
Galactose oxidase is currently used mainly for assays of D-galactose and D-galactosamine. The enzyme oxidizes the hydroxyl group in the substrate to an aldehyde, which is reactive. Therefore, the enzyme is implicated for use in production of non-natural sugars and derivatives of sugars (Arts, S. J. H. F., et al. (1997), Synthesis, June 1997, 597–613; Kosman, D. J. (1984), in Lontie, R., Eds., Copper proteins and copper enzymes, Vol. 2., CRC Press, Boca Raton, Fla., 1–26; Root, R. L., et al. (1985) J. Am. Chem. Soc. 107, 2997–2999; Mazur, A. W., and Hiler, G. D. (1997) J. Org. Chem. 62, 4471–4475.; Martin, B. D., et al. (1998), Biomaterials, 19(1–3), 69–76). The Fusarium NRRL 2903 galactose oxidase gene has been cloned (McPherson, M. J., et al. (1992), J. Biol. Chem., 267(12), 8146–8152) and expressed in Escherichia coli (Lis, M., and Kuramitsu, H. K. (1997), Antimicrob. Agents Chemother., 41(5), 999–1003).
GAO has been used in a wide variety of applications, ranging from analytical and food chemistry to chemoenzymatic synthesis and clinical testing. For example, biological sensors based on GAO have been developed to determine the content of galactose (Tkac, J., et al. (1999) Biotechnology Techniques 13, 931–936), lactose and other GAO substrates (Vega, F. A., et al. (1998) Anal. Chim. Acta 373, 57–62) in biological samples. Such biosensors have also been used for quality control in dairy industries (Adanyi, N., et al. (1999) European Food Research and Technology 209, 220–226; Mannino, S., et al. (1999) Italian Journal of Food Science 11, 57–65), online bioprocess monitoring (Szabo, E. E., et al. (1996) Biosensors & Bioelectronics 11, 1051–1058) and analysis of blood samples of patients with suspected galactosemia (Vrbova, E., et al. (1992) Collection of Czechoslovak Chemical Communications 57, 2287–2294). Additionally, GAO is also used for the detection of the disaccharide D-galactose-beta-(1→3)-N-acetylgalactosamine (Gal-GalNAc), a tumor marker in colonic cancer and precancer, and provides a cost-effective screening test for patients with neoplasia or at the risk of developing neoplasia (Yang, G. Y., and Shamsuddin, A. M. (1996) Histol. Histopathol. 11, 801–806; Said, I. T., et al., (1999) Histol. Histopathol. 14, 351–357).
GAO has also found applications in food chemistry, for example, in oxidized guar manufacture (Marrs, B. L. in IBC's Fifth Annual World Congress on Enzyme Technologies (2000) Las Vegas, Nev.) and to treat the oligosaccharide fraction contained in honey (Martin, I. G., et al. (1998) Food Chemistry 61, 281–286). Additionally, GAO has been used to oxidize the cell surface polysaccharides of membrane-bound glycoproteins containing terminal non-reducing galactose residues: this is an essential step in the successful radiolabeling of these glycoconjugates (Calderhead, D. M., and Lienhard, G. E. (1988) J. Biol. Chem. 263, 12171–12174; Gahmberg, C. G., and Tolvanen, M. (1994) Methods Enzymol. 230, 32–44).
The stereospecificity and substrate specificity of GAO have been exploited in the chemoenzymatic synthesis of L-sugars from polyols (Root, R. L., et al. (1985) J. Am. Chem. Soc. 107, 2997–2999), which are usually difficult to prepare by chemical methods (Dahlhoff, W. V., et al. (1980) Angew. Chem. Int. Ed. Engl. 19, 546–547; Koster, R., et al. (1982) Synthesis, 650–652). GAO has also been used to make sugar-containing polyamines (Liu, X. C., and Dordick, J. S. (1999) J. Am. Chem. Soc. 121, 466–467) and 5-C-(hydroxymethyl) hexoses (Mazur, A. W., and Hiler, G. D. (1997) J. Org. Chem. 62, 4471–4475).
In spite of its attractive properties and broad applicability, GAO applications in synthesis have been limited by a relatively low activity toward a large number of primary alcohols (Arts et al. (1997) Synthesis-Stuttgart 6, 597–610). The normal range of substrate specificity of GAO enzymes hampers its use for various practical applications. For example, a galactose oxidase showing activity towards new substrates such as polymeric materials and glucose would be desirable. However, previous attempts to engineer GAO to improve its activity towards D-glucose have met with difficulties, as no mutant with sufficiently improved activity towards D-glucose could be found (Sun, L., et al. (2001) Protein Eng. 14, 699–704).
Thus, there is a need to develop GAO enzymes with improved substrate specificity towards useful substrates. For example, there is a need for variant GAO enzymes with an improved ability to oxidize the 6-hydroxyl group of D-glucose. The invention addresses these and other needs in the art.